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An electron microscopic study on substance P-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs
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Brain Research, 222 (1981) 134-137 Elsevier/North-Holland Biomedical Press
An electron microscopic study on substance P-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs
HISATAKE KONDO and RYOGO YUI Department of Anatomy, Niigata University School of Medicine, Niigata, 951 (Japan) (Accepted May 28th, 1981) Key words: substance P - - celiac ganglion
guinea pig - - PAP method
Substance P-immunoreactive nerve fibers in the celiac ganglion of guinea pigs were revealed with the PAP procedures to contain abundant small clear vesicles mixed with a few large granular vesicles. The immunoreactive materials were localized around cytoplasmic components including vesicles and on the inside of the plasma membrane. The immunoreactive fibers directly apposed to unlabelled dendrites of postganglionic neurons and also to preganglionic axons. Morphological features of synapses could be identified at sites of apposition to unlabelled dendrites: clusters of vesicles in the immunoreactive fibers, intercellular spaces of about 20 nm, and an intermediate density on the postjunctional membrane of unlabelled dendrites. On the other hand, no distinct electron density together with accumulations of vesicles was seen underneath the apposed membrane of unlabelled axons. These findings indicate at the ultrastructural level that substance P-fibers form axo-dendritic synapses on the postganglionic neurons and also suggest the presence of the presynaptic interaction between substance P-fibers and some preganglionic axons in this ganglion. R e c e n t i m m u n o h i s t o c h e m i c a l studies have revealed a dense n e t w o r k o f nerve fibers with substance P-like i m m u n o r e a c t i v i t y in the p r e v e r t e b r a l ganglia including celiac a n d inferior mesenteric ganglia o f several m a m m a l s 4. The substance P - i m m u n o reactive fibers are p r e s u m e d to represent, at least in p a r t , b r a n c h e s o f p r i m a r y sensory n e u r o n s with their s o m a in the spinal ganglion3-L The i m m u n o r e a c t i v e fibers show varicosities a n d often s u r r o u n d the principal ganglion cells in a basket-like m a n n e r , suggesting t h a t these fibers m a y have f u n c t i o n a l significance in the t r a n s m i s s i o n within the ganglia. However, it c a n n o t be d e t e r m i n e d at the light m i c r o s c o p i c level w h e t h e r they exhibit a n y m o r p h o l o g i c a l features o f synapses. The present study was u n d e r t a k e n to clarify the fine structural localization o f substance P - i m m u n o r e a c t i v e nerve fibers a n d to analyze their synaptic relations to other n e u r o n a l elements in the celiac ganglion o f the guinea pig b y means o f electron microscope. Y o u n g a l b i n o guinea pigs o f b o t h sexes weighing a p p r o x i m a t e l y 250 g were used in the present study. U n d e r n e m b u t a l anesthesia (35 mg/kg) guinea pigs were perfused with 4 ~ p a r a f o r m a l d e h y d e in p h o s p h a t e buffer, p H 7.4, via the ascending a o r t a for 15 min. Celiac ganglia were then e x t i r p a t e d a n d i m m e r s e d in the same fixative for 2 h. T h e a d d i t i o n o f small percentages (0.1 ~ ) o f g l u t a r a l d e h y d e to the fixative resulted in the failure o f the detection o f the i m m u n o r e a c t i v e nerve fibers. A f t e r rinsing the tissues 0006-8993/81/0000-0000/$02.50© Elsevier/North-Holland Biomedical Press
135
Figs. 1 and 2. Substance P-like immunoreactive nerve fibers (P) have the appearance of being presynaptic to dendrites (D) of ordinary principal neurons. Note clusters of small clear vesicles mixed with a few large granular vesicles in the immunoreactive fibers and slight electron density on the postsynaptic side (arrows). x 60,000 (Fig. 1), x 33,000 (Fig. 2). Fig. 3. Direct contact (arrowhead) between a substance P-fiber (P) and a preganglionic axon (A) containing clusters of pleomorphic vesicles, x 60,000 Fig. 4. A substance P-fiber (P) and a preganglionic axon (A) form direct contact (arrowhead) and each has the appearance of being presynaptic to a dendrite (D) of an ordinary principal neuron (arrows). "< 40,000
136 with the same phosphate buffer for 12 h, sections of 20 # m thickness were cut using a Vibratome (Oxford Instrument). The sections were incubated with rabbit anti-synthetic substance P antiserum (N. Yanaihara, Shizuoka, R 400) at a dilution of 1:1000 for 12 h at 4 °C. The sites of antigen-antibody reaction were detected with the peroxidase-antiperoxidase (PAP) method according to Sternberger 1°. The control serum was obtained by the addition of 10 #g of the synthetic substance P to 1 ml of the antiserum diluted at 1:1000. After completion of the PAP procedure, the sections were fixed with l ~ osmium tetroxide in cacodylate buffer, pH 7.4, dehydrated and embedded in Epon 812. Thin sections were examined without staining or with brief staining by uranyl acetate. A total of 45 immunoreactive nerve fibers were identified in electron microscopy. They showed a varicosity ranging from 0.2 to 1.3 /~m in diameter, and were surrounded by Schwann cell and ganglionic satellite cell sheaths. The varicose fibers contained abundant small clear spherical vesicles, 45 nm in mean diameter, and a few large granular vesicles with cores showing a low electron density, 90 nm in mean diameter (Figs. 14). The low electron density of the granular vesicles may be due to the PAP procedure of the preparations. The immunoreactive materials were localized around cytoplasmic components including vesicles and mitochondria and on the inside of plasma membrane. Twenty seven of 45 labelled varicose fibers directly apposed to unlabelled neuronal profiles without intervening Schwann cell sheaths. They included 13 dendrites of postganglionic principal neurons (Figs. 1 and 2) and 14 presumably preganglionic axons (Figs. 3 and 4). The dendrites are characterized by numerous neurofilaments, neurotubules, mitochondria and few vesicles. The unlabelled axons contained abundant vesicles which consisted of small clear spherical vesicles, 45 nm in diameter and large granular vesicles with a halo, 90 nm in diameter: 4 axons contained small vesicles predominantly and 10 axons contained large vesicles more numerously. The site of apposition ranged from 0.3 to 1.0 # m in length. The apposed plasma membranes parallel to each other were separated by a space of about 20 nm. The presence of the immunoreactive material obscured the detection of any membrane specializations at the apposition site in the labeled fibers. A cytoplasmic density was slightly observed on the apposed plasma membrane of unlabeled dendrites, but no distinct electron density with the accumulation of vesicles was seen underneath the apposed membrane of unlabeled axons. No cellular components showing features of SIF (small intensely fluorescent) cells 6 were found in relation to the immunoreactive nerve fibers. After incubation with the control serum, no neuronal elements with the immunoreactivity was observed either in light microscopy or electron microscopy. The present study revealed that substance P-like immunoreactive nerve fibers form axodendritic and axoaxonic contacts. Based on the morphological criteria for chemical synapses 9, it is suggested that the axodendritic contact functions as synapses whereby the immunoreactive fibers are presynaptic. Physiological studies have demonstrated that the preganglionic stimulation elicited non-cholinergic slow EPSP in the inferior mesenteric ganglion of guinea pigs s and that the membrane depolarization
137 indistinguishable f r o m the slow E P S P was i n d u c e d b y the exogeneous a p p l i c a t i o n o f substance p1,7. I t is thus suggested t h a t substance P m i g h t be the e n d o g e n o u s transm i t t e r m e d i a t i n g this slow EPSP. The a x o d e n d r i t i c synapses revealed in the present study c o u l d be a synaptic site where substance P is released to induce this electrical potential. A t the a x o a x o n i c c o n t a c t in this m a t e r i a l it is difficult to conclude at present which one is the p r e s y n a p t i c element, because a b u n d a n t vesicles are c o n t a i n e d in b o t h n e u r o n a l profiles a n d no distinct m e m b r a n e specializations were observed at the c o n t a c t site. A previous electron m i c r o s c o p i c study with serial sections disclosed s y m m e t r i c a l m e m b r a n e specializations at the a x o a x o n i c c o n t a c t in the inferior mesenteric ganglion o f cats a n d suggested the p r e s y n a p t i c i n t e r a c t i o n between preganglionic axons at the c o n t a c t siteL W i t h this r e g a r d it should be p o i n t e d o u t t h a t the p h y s i o l o gical study d e m o n s t r a t e d the e n k e p h a l i n e r g i c p r e s y n a p t i c inhibition on the substance P - m e d i a t e d slow E P S P in the inferior mesenteric ganglion o f guinea pigs 8. Therefore, it might be possible t h a t at least some unlabeled axons c o n t a i n e n k e p h a l i n a n d exert its i n h i b i t o r y effect on the a p p o s e d i m m u n o r e a c t i v e fibers at the a x o a x o n i c contact. It is necessary to examine the fine structure o f enkephalin-like i m m u n o r e a c t i v e fibers in the ganglion. The a u t h o r s wish to t h a n k Dr. N. Y a n a i h a r a for k i n d l y p r o v i d i n g the antisubstance P a n t i s e r u m a n d Dr. T. F u j i t a for generous s u p p o r t t h r o u g h o u t this work.
1 Dun, N. J. and Karczmar, A. G., Actions of substance P in sympathetic neurons, Neuropharmacology, 18 (1979) 215-218. 2 Elfvin, L.-G., Ultrastructural studies on the synaptology of the inferior mesenteric ganglion of the cat, II. Specialized serial neuronal contacts between preganglionic end fibers, J. Ultrastruct. Res., 37 (1971) 426-431. 3 Elfvin, L.-G., Morphological studies on central and peripheral connections of sympathetic ganglia. In O. Erfink6 et al. (Eds.), Histochemistry and Cell Biology of Autonomic Neurons, SIF Cells, and Paraneurons, Raven Press, New York, 1980, pp. 335-340. 4 H6kfelt, T., Elfvin, L.-G., Schultzberg, M., Goldstein, M. and Nilsson, G., On the occurrence of substance P-containing fibers in sympathetic ganglia: Immunohistochemical evidence, Brain Research, 132 (1979) 29-41. 5 H6kfelt, T., Johansson, O., Ljungdahl, /~., Lundberg, J. M. and Schultzberg, M., Peptidergic neurons, Nature (Lond.), 284 (1980) 515-521. 6 Kondo, H., Innervation of SIF cells in the superior cervical and nodose ganglia: An ultrastructural study with serial sections, BioL Celhdaire, 30 (1977) 253 264. 7 Konishi, S., Tsunoo, A. and Otsuka, M., Substance P and non-cholinergic excitatory synaptic transmission on guinea pig sympathetic ganglia, Proc. Jap. Acad., 55 Ser. B (1979) 525-530. 8 Konishi, S., Tsunoo, A., Yanaihara, N. and Otsuka, M., Peptidergic excitatory and inhibitory synapses in mammalian sympathetic ganglia: roles of substance P and enkephalin, Biomed. Res., 1 (1980) 528-536. 9 Pappas, G. D. and Waxmen, S. G., Synaptic fine structure - - morphological correlates of chemical and electronic transmission. In G. D. Pappas and D. P. Purpura (Eds.), Structure and Function of Synapses, Raven Press, New York, 1972 pp. 1-43. 10 Sternberger, L. A., lmmunocytochemistry, Prentice-Hall, Englewood Cliffs, N. J., 1974.
Brain Research, 222 (1981) 134-137 Elsevier/North-Holland Biomedical Press
An electron microscopic study on substance P-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs
HISATAKE KONDO and RYOGO YUI Department of Anatomy, Niigata University School of Medicine, Niigata, 951 (Japan) (Accepted May 28th, 1981) Key words: substance P - - celiac ganglion
guinea pig - - PAP method
Substance P-immunoreactive nerve fibers in the celiac ganglion of guinea pigs were revealed with the PAP procedures to contain abundant small clear vesicles mixed with a few large granular vesicles. The immunoreactive materials were localized around cytoplasmic components including vesicles and on the inside of the plasma membrane. The immunoreactive fibers directly apposed to unlabelled dendrites of postganglionic neurons and also to preganglionic axons. Morphological features of synapses could be identified at sites of apposition to unlabelled dendrites: clusters of vesicles in the immunoreactive fibers, intercellular spaces of about 20 nm, and an intermediate density on the postjunctional membrane of unlabelled dendrites. On the other hand, no distinct electron density together with accumulations of vesicles was seen underneath the apposed membrane of unlabelled axons. These findings indicate at the ultrastructural level that substance P-fibers form axo-dendritic synapses on the postganglionic neurons and also suggest the presence of the presynaptic interaction between substance P-fibers and some preganglionic axons in this ganglion. R e c e n t i m m u n o h i s t o c h e m i c a l studies have revealed a dense n e t w o r k o f nerve fibers with substance P-like i m m u n o r e a c t i v i t y in the p r e v e r t e b r a l ganglia including celiac a n d inferior mesenteric ganglia o f several m a m m a l s 4. The substance P - i m m u n o reactive fibers are p r e s u m e d to represent, at least in p a r t , b r a n c h e s o f p r i m a r y sensory n e u r o n s with their s o m a in the spinal ganglion3-L The i m m u n o r e a c t i v e fibers show varicosities a n d often s u r r o u n d the principal ganglion cells in a basket-like m a n n e r , suggesting t h a t these fibers m a y have f u n c t i o n a l significance in the t r a n s m i s s i o n within the ganglia. However, it c a n n o t be d e t e r m i n e d at the light m i c r o s c o p i c level w h e t h e r they exhibit a n y m o r p h o l o g i c a l features o f synapses. The present study was u n d e r t a k e n to clarify the fine structural localization o f substance P - i m m u n o r e a c t i v e nerve fibers a n d to analyze their synaptic relations to other n e u r o n a l elements in the celiac ganglion o f the guinea pig b y means o f electron microscope. Y o u n g a l b i n o guinea pigs o f b o t h sexes weighing a p p r o x i m a t e l y 250 g were used in the present study. U n d e r n e m b u t a l anesthesia (35 mg/kg) guinea pigs were perfused with 4 ~ p a r a f o r m a l d e h y d e in p h o s p h a t e buffer, p H 7.4, via the ascending a o r t a for 15 min. Celiac ganglia were then e x t i r p a t e d a n d i m m e r s e d in the same fixative for 2 h. T h e a d d i t i o n o f small percentages (0.1 ~ ) o f g l u t a r a l d e h y d e to the fixative resulted in the failure o f the detection o f the i m m u n o r e a c t i v e nerve fibers. A f t e r rinsing the tissues 0006-8993/81/0000-0000/$02.50© Elsevier/North-Holland Biomedical Press
135
Figs. 1 and 2. Substance P-like immunoreactive nerve fibers (P) have the appearance of being presynaptic to dendrites (D) of ordinary principal neurons. Note clusters of small clear vesicles mixed with a few large granular vesicles in the immunoreactive fibers and slight electron density on the postsynaptic side (arrows). x 60,000 (Fig. 1), x 33,000 (Fig. 2). Fig. 3. Direct contact (arrowhead) between a substance P-fiber (P) and a preganglionic axon (A) containing clusters of pleomorphic vesicles, x 60,000 Fig. 4. A substance P-fiber (P) and a preganglionic axon (A) form direct contact (arrowhead) and each has the appearance of being presynaptic to a dendrite (D) of an ordinary principal neuron (arrows). "< 40,000
136 with the same phosphate buffer for 12 h, sections of 20 # m thickness were cut using a Vibratome (Oxford Instrument). The sections were incubated with rabbit anti-synthetic substance P antiserum (N. Yanaihara, Shizuoka, R 400) at a dilution of 1:1000 for 12 h at 4 °C. The sites of antigen-antibody reaction were detected with the peroxidase-antiperoxidase (PAP) method according to Sternberger 1°. The control serum was obtained by the addition of 10 #g of the synthetic substance P to 1 ml of the antiserum diluted at 1:1000. After completion of the PAP procedure, the sections were fixed with l ~ osmium tetroxide in cacodylate buffer, pH 7.4, dehydrated and embedded in Epon 812. Thin sections were examined without staining or with brief staining by uranyl acetate. A total of 45 immunoreactive nerve fibers were identified in electron microscopy. They showed a varicosity ranging from 0.2 to 1.3 /~m in diameter, and were surrounded by Schwann cell and ganglionic satellite cell sheaths. The varicose fibers contained abundant small clear spherical vesicles, 45 nm in mean diameter, and a few large granular vesicles with cores showing a low electron density, 90 nm in mean diameter (Figs. 14). The low electron density of the granular vesicles may be due to the PAP procedure of the preparations. The immunoreactive materials were localized around cytoplasmic components including vesicles and mitochondria and on the inside of plasma membrane. Twenty seven of 45 labelled varicose fibers directly apposed to unlabelled neuronal profiles without intervening Schwann cell sheaths. They included 13 dendrites of postganglionic principal neurons (Figs. 1 and 2) and 14 presumably preganglionic axons (Figs. 3 and 4). The dendrites are characterized by numerous neurofilaments, neurotubules, mitochondria and few vesicles. The unlabelled axons contained abundant vesicles which consisted of small clear spherical vesicles, 45 nm in diameter and large granular vesicles with a halo, 90 nm in diameter: 4 axons contained small vesicles predominantly and 10 axons contained large vesicles more numerously. The site of apposition ranged from 0.3 to 1.0 # m in length. The apposed plasma membranes parallel to each other were separated by a space of about 20 nm. The presence of the immunoreactive material obscured the detection of any membrane specializations at the apposition site in the labeled fibers. A cytoplasmic density was slightly observed on the apposed plasma membrane of unlabeled dendrites, but no distinct electron density with the accumulation of vesicles was seen underneath the apposed membrane of unlabeled axons. No cellular components showing features of SIF (small intensely fluorescent) cells 6 were found in relation to the immunoreactive nerve fibers. After incubation with the control serum, no neuronal elements with the immunoreactivity was observed either in light microscopy or electron microscopy. The present study revealed that substance P-like immunoreactive nerve fibers form axodendritic and axoaxonic contacts. Based on the morphological criteria for chemical synapses 9, it is suggested that the axodendritic contact functions as synapses whereby the immunoreactive fibers are presynaptic. Physiological studies have demonstrated that the preganglionic stimulation elicited non-cholinergic slow EPSP in the inferior mesenteric ganglion of guinea pigs s and that the membrane depolarization
137 indistinguishable f r o m the slow E P S P was i n d u c e d b y the exogeneous a p p l i c a t i o n o f substance p1,7. I t is thus suggested t h a t substance P m i g h t be the e n d o g e n o u s transm i t t e r m e d i a t i n g this slow EPSP. The a x o d e n d r i t i c synapses revealed in the present study c o u l d be a synaptic site where substance P is released to induce this electrical potential. A t the a x o a x o n i c c o n t a c t in this m a t e r i a l it is difficult to conclude at present which one is the p r e s y n a p t i c element, because a b u n d a n t vesicles are c o n t a i n e d in b o t h n e u r o n a l profiles a n d no distinct m e m b r a n e specializations were observed at the c o n t a c t site. A previous electron m i c r o s c o p i c study with serial sections disclosed s y m m e t r i c a l m e m b r a n e specializations at the a x o a x o n i c c o n t a c t in the inferior mesenteric ganglion o f cats a n d suggested the p r e s y n a p t i c i n t e r a c t i o n between preganglionic axons at the c o n t a c t siteL W i t h this r e g a r d it should be p o i n t e d o u t t h a t the p h y s i o l o gical study d e m o n s t r a t e d the e n k e p h a l i n e r g i c p r e s y n a p t i c inhibition on the substance P - m e d i a t e d slow E P S P in the inferior mesenteric ganglion o f guinea pigs 8. Therefore, it might be possible t h a t at least some unlabeled axons c o n t a i n e n k e p h a l i n a n d exert its i n h i b i t o r y effect on the a p p o s e d i m m u n o r e a c t i v e fibers at the a x o a x o n i c contact. It is necessary to examine the fine structure o f enkephalin-like i m m u n o r e a c t i v e fibers in the ganglion. The a u t h o r s wish to t h a n k Dr. N. Y a n a i h a r a for k i n d l y p r o v i d i n g the antisubstance P a n t i s e r u m a n d Dr. T. F u j i t a for generous s u p p o r t t h r o u g h o u t this work.
1 Dun, N. J. and Karczmar, A. G., Actions of substance P in sympathetic neurons, Neuropharmacology, 18 (1979) 215-218. 2 Elfvin, L.-G., Ultrastructural studies on the synaptology of the inferior mesenteric ganglion of the cat, II. Specialized serial neuronal contacts between preganglionic end fibers, J. Ultrastruct. Res., 37 (1971) 426-431. 3 Elfvin, L.-G., Morphological studies on central and peripheral connections of sympathetic ganglia. In O. Erfink6 et al. (Eds.), Histochemistry and Cell Biology of Autonomic Neurons, SIF Cells, and Paraneurons, Raven Press, New York, 1980, pp. 335-340. 4 H6kfelt, T., Elfvin, L.-G., Schultzberg, M., Goldstein, M. and Nilsson, G., On the occurrence of substance P-containing fibers in sympathetic ganglia: Immunohistochemical evidence, Brain Research, 132 (1979) 29-41. 5 H6kfelt, T., Johansson, O., Ljungdahl, /~., Lundberg, J. M. and Schultzberg, M., Peptidergic neurons, Nature (Lond.), 284 (1980) 515-521. 6 Kondo, H., Innervation of SIF cells in the superior cervical and nodose ganglia: An ultrastructural study with serial sections, BioL Celhdaire, 30 (1977) 253 264. 7 Konishi, S., Tsunoo, A. and Otsuka, M., Substance P and non-cholinergic excitatory synaptic transmission on guinea pig sympathetic ganglia, Proc. Jap. Acad., 55 Ser. B (1979) 525-530. 8 Konishi, S., Tsunoo, A., Yanaihara, N. and Otsuka, M., Peptidergic excitatory and inhibitory synapses in mammalian sympathetic ganglia: roles of substance P and enkephalin, Biomed. Res., 1 (1980) 528-536. 9 Pappas, G. D. and Waxmen, S. G., Synaptic fine structure - - morphological correlates of chemical and electronic transmission. In G. D. Pappas and D. P. Purpura (Eds.), Structure and Function of Synapses, Raven Press, New York, 1972 pp. 1-43. 10 Sternberger, L. A., lmmunocytochemistry, Prentice-Hall, Englewood Cliffs, N. J., 1974.